DNA vaccines provide an important new approach to vaccine development. Constructed by introducing antigen-encoding gene segments into a self-replicating plasmid of bacterial DNA, these vaccines are easy to manufacture, purify and administer. We are analyzing the nature and location of immune cells activated by DNA vaccination. Using support from the National Vaccine Program, we've also been studying the safety and mechanism of action of DNA vaccines. In research focusing on the mechanism(s) underlying DNA vaccine immunogenicity, our lab has shown that a 6 base pair DNA motif (containing an unmethylated CpG dinucleotide flanked by two 5~ purines and two 3' pyrimidines) is commonly found in DNA vaccines (derived from sequences of bacterial origin). These motifs cause the polyclonal activation of T and B lymphocytes, inducing the production of Th1 associated cytokines (such as IFNg and IL-12). This same motif contributed to the immune activation induced by DNA vaccines. Indeed, deleting these motifs (by methylation of the CpG) significantly reduced immunogenicity, whereas synthesizing new vaccines expressing additional CpG motifs improved the resultant immune response. We have further shown that co-administering these CpG motifs with conventional protein antigens has a similar effect: they significantly increase antigen-specific IgG production, and skew the immune mijlieu in favor of a Th1response. Most recently, we found that synthetic oligonucleotides expressing these CpG motifs can act as anti-allergens (by deviating the immune response towards Th1 and away from Th2 driven IgE secretion), and can be used to prevent and/or treat bacterial, parasitic and viral infections by up-regulating the innate immune system. The final aspect of this project involves the design of a DNA vaccine capable of inducing a strong immune response against HIV-derived proteins in mice. We are examining methods of improving the immunogenicity of DNA vaccines, both by inclusion of additional CpG motifs, use of lipid vehicles and gene guns for delivery, and by co-administering cytokine-encoding plasmids with the vaccine. We've found that the plasmid for GM-CSF is elicits a significant increase in cellular and humoral immunity, and improved protection in a challenge model. Finally, we've begun to explore the ability of DNA plasmids to act as gene therapy agents. We~ve shown that a plasmid encoding erythropoietin can be introduced by gene gun into the skin of normal mice, and stimulate a significant increase in serum Epo levels and hematocrit. The magnitude and duration of this effect could be regulated by the dose and frequency of plasmid administration. Since transfected skin cells are rapidly shed, safety concerns associated with the long-term transfection of cells are alleviated by this approach.